Prokaryotic culture systems such as Escherichia coli (E. coli) or other bacterial strains, which are considered fast expression systems, are most often the first choice for overproduction of proteins (Current Opinion in Biotechnology 10, 411, 1999). These systems have several known limitations, including problems in expression of large proteins, low solubility of over-expressed proteins and non-full-length gene products due to different codon usage between eukaryotes and prokaryotes, as well as lack of glycosylation. Despite these limitations, the progress made in resolving transcription, translation and protein folding processes as well as availability of improved genetic tools, make bacteria highly suitable for the expression of complex eukaryotic proteins.
Certain methods for increasing the yield of expressed proteins by variations in the culture conditions have been disclosed in yeast (EP 213 029) and in bacteria (WO 91/13156). EP 213 029 discloses the increased yield of a galactose-regulated gene product by replenishing the culture medium with fresh medium before adding the galactose to induce expression of the foreign protein. WO 91/13156 discloses the enhanced recovery of specific proteins by culturing bacterial cultures at least to the late stationary phase, without specific genetic manipulation of the regulatory genes encoded along with the recombinant protein. This disclosure neither teaches nor suggests any general mechanism for the overproduction and seems to be specific to a very restricted group of genes.
Regulation of gene expression has been found to be exerted mainly through regions of untranslated upstream DNA sequences. Deletions of material upstream or downstream from a suspected control region can be used to identify the boundaries of the promoter. Promoter regions including enhancers are characterized by their ability to bind to RNA polymerase and other activating proteins, and generally contain recognition sites for the various proteins.
One of the most essential elements is the promoter used to express the heterologous genes. U.S. Pat. No. 6,068,991 discloses a novel expression vector containing the tac promoter, based on the endogenous E. coli GroESL operon, used for over expression of heterologous genes in E. coli. Some of the other more frequently used strong promoters for the expression of heterologous genes are the promoters from PL, tac, trp, trc and the T7 promoter. The promoters used are generally regulatable. This feature is essential if the target protein to be expressed is toxic to the host. In general, the stronger the promoter, the more RNA will be transcribed from the DNA leading to the accumulation of messenger RNA. Besides strong regulatable promoters, other elements are also involved in the expression of heterologous genes. The efficiency of the translation is involved in maximizing the expression of heterologous genes. The efficiency of translation can be affected by the mRNA 5′-terminus sequences as well as by the 5′ end hairpin structure of the mRNA. Generally, a functional ribosome binding site containing a Shine-Delgarno (SD) sequence properly positioned to an AUG initiation codon is essential for efficient translation. Variation in the distance between the SD sequence and the AUG codon are known to affect mRNA translation. Studies have also shown when the SD sequence or the AUG initiation codon is sequestered in a double-stranded region of the mRNA, translation is less efficient due to the blocking of the accessibility of these sequences to the ribosome. Some other factors that have been reported to affect the efficient expression of heterologous genes are the stability of the messenger RNAs, the susceptibilities of the protein products to proteolysis and the effect of the host genetic background. Although there is a wealth of information about the elements that affect the overall efficiency of a plasmid based expression system, there are other elements that have not been studied which may be involved in the expression of heterologous genes.
Another problem presented by protein expression in prokaryotes is the three dimensional folding of proteins in an active form. Under some conditions, certain heterologous proteins expressed in large quantities from bacterial hosts precipitate within the cells in dense aggregates or inclusion bodies, and constitute a significant portion of the total cell protein. Recovery of the protein from these bodies has presented numerous problems, such as how to separate the protein encased within the cell from the cellular material and proteins harboring it, and how to recover the inclusion body protein in biologically active form. The recovered proteins are often predominantly biologically inactive because they are folded into a three-dimensional conformation different from that of the active protein. Methods for refolding the proteins into the correct, biologically active conformation are tedious, costly and time consuming, as is well known in the art.
The present invention is an unexpected result stemming from research related to structural elements that confer thermostability to bacteria. The two genes encoding for the enzyme alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobacter brockii and from the mesophile Clostridium beijerinckii (TBADH and CBADH, respectively) were isolated and compared. The genes coding for the enzymes alcohol dehydrogenase from thermophilic and mesophilic bacteria (TBADH and CBADH) have been successfully cloned, sequenced and expressed in E. coli strain TG1 using the plasmids pBS-M105/2 and pBS-P89. The level of the expressed enzymes inserted in pBluescript II KS(+) was 30-50 fold higher than in the native bacterium T. brockii or C. beijerinckii (Anaerobe 3, 259, 1997).
It was neither taught nor suggested in the background references that the overexpression attained for the thermophilic alcohol dehydrogenase could be obtained for other proteins cloned into an expression system, utilizing the bacterial ADH promoter. Furthermore, it is neither suggested nor taught that an expression system using said prokaryotic ADH promoter could yield eukaryotic heterologous proteins in their active folded form.
Fibroblast Growth Factors
Fibroblast Growth Factors (FGF's) encompass a family of at least 23 factors, which have an amino acid sequence identity ranging between 17-72%. FGF's are potent mitogens for a wide variety of cell types in tissue culture as well as in-vivo, and are regulators of differentiation of endothelial cells and neuronal cells. FGF's are expressed in a strict temporal and spatial pattern during development and have important roles in patterning and limb formation. The biological effects of FGF are initiated by binding to FGF receptors (FGFRs), phosphorylation of a trans-membrane tyrosine kinase and triggering the intracellular cascade of signal transduction.
Because of their role in growth regulation, the isolation, cloning and expression of FGF receptor ligands are extremely important for developing medicaments for the treatment of various disorders and in particular those of bone and cartilage.
Current art teaches that attempts to express synthetic DNA fragments encoding the entire reading frame of human fibroblast growth factor-1 (HBGF-1beta), and its amino terminal truncated form (HBGF-1 alpha) in Escherichia coli under the control of the trp-lac resulted in high yield and biologically active HBGF-1 alpha. (Biochem. Biophys. Acta 1090, 293, 1991) The HBGF-1 beta was highly expressed using T7 polymerase expression vector.
A synthetic gene encoding human basic FGF has been cloned and expressed in E. coli as a biologically active protein using the expression vector pLCII downstream from the strong PL promoter (J. Biotechnol. 22, 299, 1992).
There is an unmet need for and it would be advantageous to have a high yield expression system that can be used to express various FGF agonists, FGF antagonists, FGF mutants or FGF chimeras having therapeutic value for treating various diseases.